Abstract Strategies to restore function in damaged hearts often focus on the replacement of lost cardiomyocytes (CMs). Depending on the approach, the new CMs may be derived through the differentiation of exogenous pluripotent stem cells, the differentiation of resident progenitors, the proliferation of endogenous CMs, or reprogramming of non-CMs to CMs. Unfortunately, CMs generated by these approaches have thus far made poor substitutes for mature myocardium, with deficiencies in both electrical and mechanical function. Studies indicate that CMs from regenerative strategies often resemble CMs of the fetal or neonatal heart, rather than the adult, making CM maturation a major roadblock in the field. This obstacle has been difficult to overcome due to insufficient knowledge of how CM maturation is transcriptionally regulated. Here we propose addressing this deficiency by first utilizing an established genetic model of a CM growth defect to identify and analyze factors that regulate CM maturation. Second, we will combine insights gained from this model with a novel technical approach to screen for novel transcriptional regulators of CM maturation in vivo. GATA4 and GATA6 are zinc finger transcription factors that play key roles in cardiac function and development. Mosaic double knockout of myocardial GATA4/6 via low dose administration of AAV9-TNT-Cre to neonatal mice intriguingly appears to result in stalled CM maturation. By adulthood, GATA4/6 mutant cells are dramatically smaller than their Cre- counterparts, resembling neonatal CMs. As GATA4/6 proved to be crucial and redundant regulators of CM growth, we reasoned that analysis of GATA4 targets is likely to reveal factors that mediate CM maturation. Therefore, we conducted neonatal GATA4 ChIP-seq to identify likely regulators of CM maturation. In Aim 1 we outline a strategy to functionally analyze the role of two promising candidates in vivo during neonatal CM maturation. These factors, Fhod3 and Daam1, belong to the Formin family of actin binding proteins, which have previously been linked to sarcomere assembly and maintenance. The striking phenotype of GATA4/6 mutant CMs indicated that transcriptional regulators of CM maturation can be identified by assessing cell size in a mosaic loss-of-function model. However screening factors in vivo one at a time is prohibitively costly. In Aim 2 we propose an in vivo screen that will utilize cutting edge CRISPR technologies to allow many genes to be tested in a single animal. This screen will use the cell autonomous effect of gene knockout on individual CM growth as the readout. This unbiased approach will be used to discover new transcriptional regulators of neonatal CM growth, which is a hallmark of CM maturation. Successful completion of this Aim will allow us to use candidates as new genetic entry points, which can be exploited by ChIP-seq and RNA-seq to rapidly dissect the transcriptional network that governs neonatal CM growth and maturation. Collectively, the complementary approaches of Aims 1 and 2 will greatly increase our knowledge of this maturation network, and will have the potential to enhance regenerative therapeutic strategies.